Most T lymphocytes recognize peptides presented by MHC-encoded class I and class II molecules, but recently it has been shown that some T cells can respond either to glycopeptides or to glycolipids. In such cases, the exposed carbohydrates are believed to interact with the T cell antigen receptor (TCR), leaving the lipid or peptide portion buried in the antigen presenting molecule. The long-term goal of this application is to achieve a better understanding of the structural basis for the TCR-mediated recognition of carbohydrate containing antigens. In particular, we will address the following questions: 1. What is the size of the carbohydrate that can be recognized by the TCR? 2. How discriminating is the TCR in distinguishing between closely related carbohydrates? 3. What are the affinity and the kinetics of the TCR interaction with carbohydrates? 4. To what degree can carbohydrate antigens be processed or catabolized for recognition? While we have expertise in molecular biology and cellular immunology, the resources and expertise provided by the cores will give us the ability to analyze these issues at the biochemical level, with a degree of detail and precision that would not otherwise be possible. As a model system to study T cell responses to carbohydrate containing antigens, we will characterize the recognition of the lipoglycan alpha- galactosyl ceramide (alphaGalCer). This recognition only occurs when alphaGalCer is bound to or presented by CD1d, a non-classical class I molecule. The responding T lymphocytes are a well-defined subset, known as NK T cells. Using alpha GalCer analogs synthesized by Core D (carbohydrate synthesis and protein expression) and soluble CD1d molecules also produced by this core, we will determine if carbohydrates influence the binding to CD1d, as well as the nature of the carbohydrate interaction with the TCR of NK T cells. Recognition will be assessed using both immune response assays and in vitro binding assays of soluble TCRs (produced in core D) to soluble recombinant CD1d molecules that have been preloaded with the appropriate antigens. These measurements will be made by Core K (Protein-Carbohydrate interaction core). We will analyze whether T cell recognition requires internalization of antigen by the CD1d expression antigen presenting cell (APC), and removal of some of the terminal saccharide groups, and if so, where in the cell this occurs. This will be done using the techniques of cell biology as well as analytical chemistry, carried out in part by Core C (Analytical Glycotechnology). By adding specific enzyme inhibitors, or by using cells from mice deficient for genes involved in glycolipid catabolism, we will identify the enzymes responsible for the processing or catabolism of this unique class of antigens.